To further reveal the effect of CR and mimetics on protein biosynthesis and degradation, we examined the expression levels of yeast ribosomal protein large subunit genes (Rpl) and ribosomal protein small subunit genes (Rps) after treatment by CR and mimetics. As the consequence, more Rpl/Rps and other related genes are downregulated by CR and mimetics. During treatment, a dramatic decreased protein concentration was noticed in all tested samples. These results indicated that the biosynthesis of ribosomal proteins is inhibited, and ART or H₂O₂ can simulate CR in the modulation of ribosome genes and hence their expression.

Accordingly, ribosome biogenesis genes that accelerate the ribosomal protein biosynthesis were found to be highly upregulated in all groups, implying a feedback response of ribosomes from various stress conditions. Not surprisingly, upregulation of ribosome biogenesis genes is more remarkable for the CR group because its ribosome genes are extremely downregulated. At the same time, we also analyzed the impact of different treatments on the controlled degradation of proteins through ubiquitylation. Consequently, some ubiquitylation genes (Ubi) are upregulated, but others downregulated. These results proved that selective protein degradation is enhanced after treatments, by which amino acids that are released from degraded proteins can be re-utilized. Furthermore, it was also found that most of autophagy genes (Atg) are upregulated by each treatment albeit a few of which are downregulated. It is clear that CR and mimetics can partially augment autophagy in order to help yeast coping with environmental stress (Fig. 6.1).

Genome-wide expression profiling was used in the present study to compare the effects of CR and mimetics on ATP-binding cassette transporter gene expression profiles and lipid metabolic patterns. The dynamic changes of SOD activity were monitored in pretreated and post-treated yeast cells. After treatments, cellular detoxification-related ATP-binding cassette transporter genes are downregulated or unchanged, whereas the peroxisomal ATP-binding cassette transporter genes Pxa1 and Pxa2 that enhance long-chain fatty acid transport as well as the plasma membrane ATP-binding cassette transporter genes Aus1 and Pdr11 that accelerate sterol uptake are considerably upregulated (Table 6.2).

These results indicated that mitohormesis depends on ATP-binding cassette transporter-mediated lipid transport and subsequent lipid degradation and re-utilization (Wang and Zeng 2014).

The metabolic flux of glucose flows from the cytosol to mitochondria. Because the cytosolic glycolysis pathway genes are globally downregulated by CR and mimetics, it could be predicted that the mitochondrial oxidative phosphorylation pathway genes should be accordingly downregulated by each treatment. Indeed, it was found that those genes encoding COX (complex IV), F₁F₀-ATP synthase (Complex V), and other associated signature proteins are downregulated in every group, although the expression levels of those genes are much lower in the CR group than in the mimetic groups.

The analytic data of transcriptomic microarray indicated that H₂O₂ degradation-responsible antioxidant genes, such as Cat1 encoding mitochondria-localized CAT and Gpx2 encoding cytosolic GSH-POX, are upregulated by CR and mimetics. In contrary, H₂O₂ generation-responsible antioxidant genes, such as Sod1 and Sod2 coding for cytosolic copper-zinc SOD (Cu/Zn-SOD) and mitochondrial manganese SOD (Mn-SOD), are downregulated by CR and mimetics, implying that the main type of ROS in this stage is H₂O₂ other than O₂ ⁻. Besides, it was also seen that other antioxidant enzyme-encoding genes are either upregulated (Trx3), or downregulated (Gto3 and Dot5), or unchanged in different treatment groups.

As experimental evidence supporting above suggestion of H₂O₂ as a main type of ROS, we investigated the expression modes and activity dynamics of mitochondrial signatures including antioxidant enzymes. In ME yeast cells incubated for 12 h, CR increases both Cu/Zn-SOD and Mn-SOD activities. In contrast, in PME yeast cells incubated for several days, CR decreases both Cu/Zn-SOD and Mn-SOD activities. So CR-based “dual-phase responses” can be typically distinguished by the “up-and-down” modes of antioxidant responses.

Additionally, an elevation of the expression level of peroxisome genes in all treatment groups seemed to support above deduction that H₂O₂ has become the major kinds of ROS, which can be thoroughly scavenged by the cooperation of CAT, GSH-POX, and peroxisomal enzymes. More importantly, upregulation of peroxisomal β-oxidation enzymes contributes to energy generation by the oxidation of fatty acids released by the degraded storage lipids and recycled cellular components during carbon starvation.

It is noted that a repression of the respiratory activity should lead to the decrease of ATP and NADH, i.e., the elevation of AMP/ATP and NAD⁺/NADH ratios, which can in turn activate AMPK and SIRT1 separately. In postlogarithmic yeast cells, only a slight upregulation of Snf1 (coding for a yeast homolog of AMPK) and Sir2 (coding for a yeast homolog of SIRT1) was observed in CR treatment, but no significant changes of those genes were noticed in mimetic treatment. Additionally, Sch9, encoding a yeast homolog of mammalian ribosomal S6 kinase 1 (S6K1), was also found to be weakly upregulated in the CR group but unchanged in mimetic groups.

It was shown that mitogen-activated protein kinase (MAPK) pathway genes (Kss1 and Fus3 are yeast homologs of MAPK) are mostly downregulated by CR and mimetics, indicating a common modulation pattern in the PE phase. As a critical gene controlling cell fate and determining lifespan, Tor is upregulated in either treatment group, in which Tor1 is considerably upregulated for many folds. Because TOR is activated via phosphorylation, we do not expect that TOR activity is correlated with its transcript abundance. Nevertheless, it seemed that the inhibition of TOR activity may induce Tor expression perhaps in a feedback manner.

We further quantified the induced expression levels of Snf1, Tor1, and Kss1 in logarithmic yeast among treatment groups. It is very clear that their expression patterns are distinct in the logarithmic stage from those in the postlogarithmic stage. For example, Tor1 exhibits downregulation in the logarithmic stage, but shows upregulation in the postlogarithmic stage. In the logarithmic yeast, the overall expression levels of Snf1 and Kss1 that are induced by CR, ART, or H₂O₂ are typically lower than those in calorie nonrestriction. These results further strengthened that protein kinase genes are regulated in a dual-phase mode.